Selection and Use of Lactic Acid Bacteria Preventing Bone Loss in Mammals

ABSTRACT

The present invention comprises a method for selecting lactic acid bacterial strains effective for preventing bone loss in humans and strains that have been selected according to the presented method. The selection method is based on the strains capability of reestablishing an altered microbial community to normal and/or harboring at least one of four specific SNPs.

STATEMENT OF PRIORITY

This application is a continuation application and claims priority toU.S. patent application Ser. No. 15/918,207, filed Mar. 12, 2018, whichis a divisional application of Ser. No. 14/405,496, filed Dec. 4, 2014,which claims the benefit of International Application Serial No.PCT/SE2013/050646, filed Jun. 4, 2013, which claims the benefit, under35 U.S.C. § 119 (a) of U.S. Provisional Patent Application Ser. No.61/689,338, filed Jun. 6, 2012, the entire contents of each of which areincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to medicine, pharmacology andfood supplements. More specifically the invention relates to selectionand use of lactic acid bacteria for the prevention of bone loss inmammals.

BACKGROUND OF THE INVENTION

Over 40 million Americans over the age of 50 (14 million of which aremen) are afflicted with low bone density or osteoporosis and itsassociated increased risk of fractures. Individuals with osteoporoticfractures are prone to depression, dependency and increased mortality.While aging is a major cause of osteoporosis, disease, disuse, andcertain drugs can also cause bone loss at any stage in life.

The skeleton is a highly organized system that supports the body'sweight, houses mesenchymal and hematopoetic stem cells, and serves as acalcium reservoir. The structure of bone comprises an outer corticaldense shell and an inner trabecular bone meshwork. Exercise can increasetrabecular bone mineral density (BMD), and bone volume fraction (BVF),trabeculi thickness, and cortical BMD and thickness. In contrast,disease, disuse, and certain drugs (such as glucocorticoids) candecrease these parameters and cause osteoporosis in both males andfemales. Osteoporosis is defined by a reduction in bone mass (more than2.5 standard deviations (SD) below average) and altered bonemicro-architecture (such as decreased trabeculi thickness). Withdecreasing bone mass there is an increased risk of bone fractures. Thus,at the point of being diagnosed as osteoporotic, a patient has a 16-foldincrease in fracture risk compared to someone with normal bone density.Fractures are associated with depression, dependency, and increasedmortality (greater than 25% within 12 months for the elderly) and hipfractures account for over 50,000 deaths annually (National OsteoporosisFoundation (NOF) statistic). While osteoporosis is less prevalent inmen, over 30% of hip fractures occur in men and mortality rates aregreater for males compared to females. Currently, over 20 billiondollars are spent in the US and 30 billion dollars in the European Unionto cover the direct costs of osteoporosis. Of even greater concern, itis estimated that by 2020 more than 61 million men and women in the US,over the age of 50, will have low bone density or osteoporosis (NOFstatistic), and finding effective novel treatments is therefore apriority. In fact, one in three women over the age of 50 will experiencean osteoporosis related fracture in their lifetime. Along with itsassociated increase in fracture risk, bone loss may have negativeeffects on metabolism and insulin secretion. Despite all the availabletreatments on the market, the number of osteoporotic patients is on therise in the U.S. and worldwide. There are several reasons for this,including a lack of awareness that one is at risk early in life, anincreasing elderly population, and patient noncompliance due to unwantedmedication side effects. In addition, conventional bone loss treatmentsare not always effective. Currently there are no alternative or naturaltreatments that can be used in place of osteoporosis medicines forpeople with low bone density or osteoporosis. Therefore, doctors arelooking for new approaches to increase bone density in their patientsand companies are working to improve pharmacologic bone therapeuticdrugs.

Certain people are more likely to develop osteoporosis than others, somerisk factors are;

-   -   Being female    -   Older age    -   Family history of osteoporosis or broken bones    -   Being small and thin    -   Certain race/ethnicities such as Caucasian, Asian, or        Hispanic/Latino although African Americans are also at risk    -   History of broken bones    -   Low levels of sex hormones    -   Low estrogen levels in women, including menopause    -   Missing periods (amenorrhea)    -   Low levels of testosterone and estrogen in men    -   Diet        -   Low calcium intake        -   Low vitamin D intake        -   Excessive intake of protein, sodium and caffeine    -   Inactive lifestyle    -   Smoking    -   Alcohol abuse    -   Certain medications such as steroid medications, some        anticonvulsants and others    -   Certain diseases and conditions such as anorexia nervosa,        rheumatoid arthritis, gastrointestinal diseases and others

Menopausal women are prone to losing bone during menopause time due todecreased estrogen levels. Even during perimenopause (the period of 2 to8 years before menopause) estrogen levels may start to drop off. Overtime, too much bone loss can first cause osteopenia (low bone mass) andthen osteoporosis.

Diagnosis of type 1 diabetes (T1D) is increasing in children and adults.While medical advances are extending patient lifespan, maintainingeuglycemia remains difficult, even under therapeutic vigilance. Thus,more T1D patients (males and females) are suffering from complications,including bone loss. This means that patients begin aging/menopause withan already increased fracture risk. Once fractures occur, they can bedifficult to heal, require extended hospitalizations, reduce the qualityof life and increase mortality. Poor bone health also negatively affectsthe entire body. Postmenopausal women with T1D diabetes have higherincidences of osteoporotic fractures than women without diabetes.Children with T1D have lower bone mineral density than children withoutdiabetes. Thus, maintaining bone health is critical for the overallquality of life of T1D patients and important for maximizingtherapeutic/curative treatments involving marrow immune/progenitor cellssince marrow cells and bone cells communicate.

Type 2 Diabetes (T2D) patients are also at higher risk of osteoporoticfractures than non-diabetics.

The two key components to strengthening bone and preventing osteoporosisare 1) attaining maximum bone density and 2) preventing bone loss duringadulthood and aging. Bone remodeling occurs because bone is dynamic andconstantly adapts to environmental cues to form or resorb bone. Targetedbone remodeling through the activities of osteoblasts (bone formingcells) and osteoclasts (bone resorbing cells) maintains blood calciumlevels within a critical range while keeping bone strong at sites wheresupport is needed. When formation and resorption activities are inbalance there is no net gain or loss of bone, however when formation isdecreased and/or resorption is increased then bone loss ensues.

Increased osteoclast activity results in bone resorption. Osteoclastsare derived from hematopoetic stem cells. These cells give rise to cellsof the monocyte/macrophage lineage which, under the right conditions,develop into osteoclast precursors. Further signaling through factorssuch as RANKL (located on osteoblast surfaces) stimulate osteoclastmaturation. Mature osteoclasts express enzymes involved in bone matrixdegradation (including cathepsin K and TRAP5b).

Increased osteoblast activity results in bone formation, which can beregulated at several levels including 1) lineage selection, 2)maturation and 3) death. Because bone marrow stromal cells (BMSC) giverise to osteoblasts, adipocytes and other cell types, selection of onelineage (adipocyte) could be at the cost of another (osteoblast). Thisis supported by the reciprocal relationship between bone adiposity andmineral density recognized with aging, limb unloading, cell culturemodels, and type I (T1) diabetes. Osteoblast activity can be furtherregulated through death/apoptosis. An increase in osteoblast death willresult in fewer bone making cells and therefore bone loss. Examplesinclude the rapid bone adaptation to disuse/unloading, which results inbone loss, increased marrow adiposity, and increased bone cell death.Aging also increases bone cell apoptosis. Many factors contribute tomodulating some or all aspects of osteoblast regulation (lineage,maturation, death) including: positive factors such as TGFβ, bonemorphogenic proteins (BMPs), parathyroid hormone (PTH), and Wnts andnegative factors such as cytokines.

Bisphosphonates are one of the most common treatments for osteoporosis.These compounds incorporate into the bone mineral and inhibit bonecatabolism by osteoclasts and are effective at reducing fractures.However, many of these compounds need to be taken on an empty stomachand can cause gastric reflux and nausea resulting in reduced patientcompliance. There is also concern about the length of time that thesecompounds reside in bone and their long-term impact on bone remodelingand strength. Selective estrogen receptor modifiers (SERMS) are anothertherapeutic treatment, but they still carry some concerns with regard tocancer. Hormone replacement therapy has been studied as useful inpreventing or slowing the occurrence of osteoporosis, but sustained useof hormone replacement over many years may increase women's risk ofbreast cancer, may increase incidence of venous thrombosis (bloodclots), exacerbation of pre-existing liver diseases and an increasedrisk of endometrial cancer as well as hypertension. Amgen has a drugunder development (that is similar to osteoprotegrin) that works bymodifying the RANKL/RANK system and hence suppresses osteoclastactivity. Intermittent PTH treatment is an anabolic treatment, but thisintravenous treatment is expensive and only indicated for severeosteoporotic patients. Taken together, it is not surprising that manypeople diagnosed with low bone density are confused about what to do.Many people do not want to take medication for fear of long-termeffects. While weight bearing exercise and adequate calcium intake aretwo natural approaches, they cannot always overcome effects of disease,medications, and aging.

SUMMARY OF THE INVENTION

A primary object of the present invention is to provide a method how tofind a lactic acid bacterial strain that may prevent bone loss,especially in menopausal women, in diabetics, in osteopenic thatincludes for example young men with large energy intake and low exercisefrequency.

An object of the present invention is to use products containing suchstrains in menopausal women to prevent bone loss.

An object of the present invention is to use products containing suchstrains in women who have had a hysterectomy to prevent bone loss.

Another object is to use products containing such strains in men,including but not limited to diabetic, young men with metabolicdisturbance and osteopenic men, to prevent bone loss.

Another object is to use such product in combination with therapies forbone loss or bone formation in order to reduce the dose of such drugs tobe able to minimize side effects.

Another object is to improve bone repair after fracture.

Accordingly, a first aspect of the invention provides a method forselecting a lactic acid bacterial strain for use in preventing ortreating bone loss, comprising selecting a lactic acid bacterial strainhaving at least 95% identity to the genome of L reuteri JCM 1112 (SEQ IDNO: 1), and harboring an identical nucleotide relative to the genome ofL reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following fourpositions: C in base pair 271 391, G in base pair 453 538, G in basepair 529 228, and C in base pair 599 338.

In an embodiment according to the first aspect, the method comprisesselecting a lactic acid bacterial strain having at least 96%, such as97%, such as 98%, such as 99% identity to the genome of L reuteri JCM1112 (SEQ ID NO: 1), and harboring an identical nucleotide relative tothe genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of thefollowing four positions: C in base pair 271 391, G in base pair 453538, G in base pair 529 228, and C in base pair 599 338.

A second aspect of the invention provides a method for selecting alactic acid bacterial strain, such as a Lactobacillus reuteri strain,for use in preventing or treating bone loss, comprising selecting aLactobacillus reuteri harboring an identical nucleotide relative to thegenome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of thefollowing four positions: C in base pair 271 391, G in base pair 453538, G in base pair 529 228, and C in base pair 599 338.

In an embodiment of the method according to the first or second aspect,the lactic acid bacterial strain harbors at least two of said fournucleotides, such as at least three of said four nucleotides, such asall four of said nucleotides.

A third aspect of the invention provides a method for selecting a lacticacid bacterial strain for use in preventing or treating bone loss,comprising selecting a lactic acid bacterial strain having at least 95%identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), providedthat the lactic acid bacterial strain does not harbor at least onemutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1),selected from the group of four mutations consisting of C to T in basepair 271 391, G to A in base pair 453 538, G to A in base pair 529 228,and C to T in base pair 599 338.

In an embodiment of the third aspect, the method comprises selecting alactic acid bacterial strain having at least 96%, such as 97%, such as98%, such as 99% identity to the genome of L reuteri JCM 1112 (SEQ IDNO: 1), provided that the lactic acid bacterial strain does not harborat least one mutation relative to the genome of L reuteri JCM 1112 (SEQID NO: 1), selected from the group of four mutations consisting of C toT in base pair 271 391, G to A in base pair 453 538, G to A in base pair529 228, and C to T in base pair 599 338.

In an embodiment of the method according to the third aspect, the lacticacid bacterial strain does not harbor at least two of said fourmutations, such as at least three of said four mutations, such as anyoneof said four mutations.

A fourth aspect of the invention provides a lactic acid bacterialstrain, selected according to the method according to the first, secondor third aspect, for use in the prevention or treatment of bone loss.

In an embodiment of the fourth aspect, the lactic acid bacterial strainselected is L reuteri ATCC PTA 6475, for use in the prevention ortreatment of bone loss. This strain is available to the public at theAmerican Type Culture Collection (10801 Univ. Blvd., Manassas, Va.),having been deposited there under the Budapest Treaty on Dec. 21, 2004.

According to a fifth aspect, the invention provides a lactic acidbacterial strain having at least 95% identity to the genome of L reuteriJCM 1112 (SEQ ID NO: 1), and harboring an identical nucleotide relativeto the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one ofthe following four positions: C in base pair 271 391, G in base pair 453538, G in base pair 529 228, and C in base pair 599 338, for use in theprevention or treatment of bone loss.

In an embodiment of the fifth aspect, the lactic acid bacterial strainhas at least 96%, such as 97%, such as 98%, such as 99% identity to thegenome of L reuteri JCM 1112 (SEQ ID NO: 1), and harbors an identicalnucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1)in at least one of the following four positions: C in base pair 271 391,G in base pair 453 538, G in base pair 529 228, and C in base pair 599338.

In an embodiment of the fifth aspect, the lactic acid bacterial strainharbors at least two of said four nucleotides, such as at least three ofsaid four nucleotides, such as all four of said nucleotides.

According to a sixth aspect, the invention provides a lactic acidbacterial strain having at least 95% identity to the genome of L reuteriJCM 1112 (SEQ ID NO: 1), provided that the lactic acid bacterial straindoes not harbor at least one mutation relative to the genome of Lreuteri JCM 1112 (SEQ ID NO: 1), selected from the group of fourmutations consisting of C to T in base pair 271 391, G to A in base pair453 538, G to A in base pair 529 228, and C to T in base pair 599 338.

In an embodiment of the sixth aspect, the lactic acid bacterial strainhas at least 96%, such as 97%, such as 98%, such as 99% identity to thegenome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lacticacid bacterial strain does not harbor at least one mutation relative tothe genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the groupof four mutations consisting of C to T in base pair 271 391, G to A inbase pair 453 538, G to A in base pair 529 228, and C to T in base pair599 338.

In an embodiment of the sixth aspect, the lactic acid bacterial straindoes not harbor at least two of said four mutations, such as at leastthree of said four mutations, such as anyone of said four mutations.

According to a presently preferred embodiment of the fifth or sixthaspect, the lactic acid bacterial strain is L reuteri ATCC PTA 6475.

A seventh aspect of the invention provides a composition comprising alactic acid bacterial strain selected according to the method accordingto the first, second, or third aspect of the invention.

According to an eighth aspect, a composition is provided comprising alactic acid bacterial strain having at least 95% identity to the genomeof L reuteri JCM 1112 (SEQ ID NO: 1), and harboring an identicalnucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1)in at least one of the following four positions: C in base pair 271 391,G in base pair 453 538, G in base pair 529 228, and C in base pair 599338.

In an embodiment of the eighth aspect, the lactic acid bacterial strainhas at least 96%, such as 97%, such as 98%, such as 99% identity to thegenome of L reuteri JCM 1112 (SEQ ID NO: 1), and harbors an identicalnucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1)in at least one of the following four positions: C in base pair 271 391,G in base pair 453 538, G in base pair 529 228, and C in base pair 599338.

In an embodiment of the eighth aspect, the lactic acid bacterial strainharbors at least two of said four nucleotides, such as at least three ofsaid four nucleotides, such as all four of said nucleotides.

According to a ninth aspect, the invention provides a compositioncomprising a lactic acid bacterial strain having at least 95% identityto the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that thelactic acid bacterial strain does not harbor at least one mutationrelative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selectedfrom the group of four mutations consisting of C to T in base pair 271391, G to A in base pair 453 538, G to A in base pair 529 228, and C toT in base pair 599 338.

In an embodiment of the ninth aspect, the lactic acid bacterial strainhas at least 96%, such as 97%, such as 98%, such as 99% identity to thegenome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lacticacid bacterial strain does not harbor at least one mutation relative tothe genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the groupof four mutations consisting of C to T in base pair 271 391, G to A inbase pair 453 538, G to A in base pair 529 228, and C to T in base pair599 338.

In an embodiment of the ninth aspect, the lactic acid bacterial straindoes not harbor at least two of said four mutations, such as at leastthree of said four mutations, such as anyone of said four mutations.

In a presently preferred embodiment of the eighth or ninth aspect, thelactic acid bacterial strain is L reuteri ATCC PTA 6475.

In an embodiment of the eighth or ninth aspect, the composition is foruse in the prevention or treatment of bone loss.

In another embodiment of the eighth or ninth aspect, the composition isfor use in preventing bone loss in menopausal women, women who have hadhysterectomy, diabetics, osteopenic individuals, osteoporoticindividuals, and individuals with metabolic disturbance.

In yet another embodiment of the eighth or ninth aspect, the compositionis for use in improving bone repair after fracture.

In an embodiment of the eighth or ninth aspect, the above-describedcomposition in combination with vitamin D is for use in preventing ortreating bone loss.

In another embodiment of the eighth or ninth aspect, the above-describedcomposition in combination with a hormone (for use in hormonereplacement therapy) is for use in preventing or treating bone loss.

In an embodiment of the eighth or ninth aspect, the above-describedcomposition is a pharmaceutical composition (optionally comprising atleast one pharmaceutically acceptable excipient), or a food product or afood supplement (optionally comprising at least one food-gradeexcipient, as known to a person of ordinary skill in the art).

According to a tenth aspect, the invention provides a use of a lacticacid bacterial strain having at least 95% identity to the genome of Lreuteri JCM 1112 (SEQ ID NO: 1), and harboring an identical nucleotiderelative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at leastone of the following four positions; C in base pair 271 391, G in basepair 453 538, G in base pair 529 228, and C in base pair 599 338, forthe manufacture of a pharmaceutical composition for the prevention ortreatment of bone loss.

In an embodiment of the tenth aspect, the lactic acid bacterial strainhas at least 96%, such as 97%, such as 98%, such as 99% identity to thegenome of L reuteri JCM 1112 (SEQ ID NO: 1), and harbors an identicalnucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1)in at least one of the following four positions: C in base pair 271 391,G in base pair 453 538, G in base pair 529 228, and C in base pair 599338.

In an embodiment of the tenth aspect, the lactic acid bacterial strainharbors at least two of said four nucleotides, such as at least three ofsaid four nucleotides, such as all four of said nucleotides.

According to an eleventh aspect, the invention provides a use of alactic acid bacterial strain having at least 95% identity to the genomeof L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactic acidbacterial strain does not harbor at least one mutation relative to thegenome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the group offour mutations consisting of C to T in base pair 271 391, G to A in basepair 453 538, G to A in base pair 529 228, and C to T in base pair 599338, for the manufacture of a pharmaceutical composition for theprevention or treatment of bone loss.

In an embodiment of the eleventh aspect, the lactic acid bacterialstrain has at least 96%, such as 97%, such as 98%, such as 99% identityto the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that thelactic acid bacterial strain does not harbor at least one mutationrelative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selectedfrom the group of four mutations consisting of C to T in base pair 271391, G to A in base pair 453 538, G to A in base pair 529 228, and C toT in base pair 599 338.

In an embodiment of the eleventh aspect, the lactic acid bacterialstrain does not harbor at least two of said four mutations, such as atleast three of said four mutations, such as anyone of said fourmutations.

In a presently preferred embodiment of the tenth or eleventh aspect, thelactic acid bacterial strain is L reuteri ATCC PTA 6475.

A twelfth aspect of the invention provides a method for the treatment orprevention of bone loss, comprising administering, to an individual, alactic acid bacterial strain having at least 95% identity to the genomeof L reuteri JCM 1112 (SEQ ID NO: 1), and harboring an identicalnucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1)in at least one of the following four positions: C in base pair 271 391,G in base pair 453 538, G in base pair 529 228, and C in base pair 599338.

In an embodiment of method according to the twelfth aspect, the lacticacid bacterial strain has at least 96%, such as 97%, such as 98%, suchas 99% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), andharbors an identical nucleotide relative to the genome of L reuteri JCM1112 (SEQ ID NO: 1) in at least one of the following four positions: Cin base pair 271 391, G in base pair 453 538, G in base pair 529 228,and C in base pair 599 338.

In an embodiment of the twelfth aspect, the lactic acid bacterial strainharbors at least two of said four nucleotides, such as at least three ofsaid four nucleotides, such as all four of said nucleotides.

According to a thirteenth aspect, the invention provides a method forthe treatment or prevention of bone loss, comprising administering, toan individual, a lactic acid bacterial strain having at least 95%identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), providedthat the lactic acid bacterial strain does not harbor at least onemutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1),selected from the group of four mutations consisting of C to T in basepair 271 391, G to A in base pair 453 538, G to A in base pair 529 228,and C to T in base pair 599 338.

In an embodiment of the thirteenth aspect, the lactic acid bacterialstrain has at least 96%, such as 97%, such as 98%, such as 99% identityto the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that thelactic acid bacterial strain does not harbor at least one mutationrelative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selectedfrom the group of four mutations consisting of C to T in base pair 271391, G to A in base pair 453 538, G to A in base pair 529 228, and C toT in base pair 599 338.

In an embodiment of the thirteenth aspect, the lactic acid bacterialstrain does not harbor at least two of said four mutations, such as atleast three of said four mutations, such as anyone of said fourmutations.

In a presently preferred embodiment of the twelfth or thirteenth aspect,the lactic acid bacterial strain is L reuteri ATCC PTA 6475.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows microbial community clustering in jejunum and ileum.

FIG. 2 shows the suppression of bone loss by L. reuteri ATCC PTA 6475.

FIG. 3 shows the effect on bone loss in different L. reuteri strains.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

Chronic inflammatory diseases are frequently associated with systemicbone loss. In the NIH grant abstract, grant number 1R21AT005472-01A1,McCabe suggests that therapies which improve overall intestinal healthhave the potential to benefit bone health. McCabe and Britton found outthat L. reuteri treatment decreased TNF levels in the ileum andincreased bone volume in healthy male but not female mice and suggestthat L. reuteri increases bone density in a gender dependent mannerthrough suppression of intestinal inflammation and upregulation of boneformation. They suggest that they have a novel way of increasing bonemass by use of a probiotic bacterium that attenuates intestinalinflammation. This is a gender dependent up regulation of boneformation, not prevention of bone loss, associated to L. reuteri'santi-inflammatory properties, unlike the present invention where certainspecifically selected strains are used to prevent bone loss in both maleand female.

Probiotics can increase chicken cortical bone thickness and reduce boneloss in aging mice. Narva et al. described in “Effects of bioactivepeptide, valyl-prolyl-proline (VPP), and lactobacillus helveticusfermented milk containing VPP on bone loss in ovariectomized rats.” thatL. helveticus fermented milk prevent bone loss, the effect might be dueto the peptide valyl-prolyl-proline. Narva et al. further described in“The effect of Lactobacillus helveticus fermented milk on acute changesin calcium metabolism in postmenopausal women” that fermentation of milkwith Lactobacillus helveticus had a positive acute effect on calciummetabolism.

Yeo et al. suggest in “Angiotensin I-converting enzyme inhibitoryactivity and bioconversion of isoflavones by probiotics in soymilksupplemented with prebiotics” that probiotic incorporated into soymilksupplemented with prebiotic could potentially be used as a dietarytherapy in for example osteoporosis.

Kim et al. showed in “Effects of a Lactobacillus casei 393 fermentedmilk product on bone metabolism in ovariectomised rats” that L. casei393 FMP had a preventative effect on bone loss in ovariectomised rats.

However none of the above mentioned prior art neither alone nor incombination teaches how one can select specific probiotic strains thatare effective for preventing bone loss.

The present invention herein comprises a method for selecting lacticacid bacterial strains effective for preventing bone loss in humans andstrains that have been selected according to the presented method.Products such as foods, nutritional additives and formulations,pharmaceuticals or medical devices containing whole cells or componentsderived from these strains may be formulated as is known in the art, andgenerally include an ingestible support as known plus the lactic acidbacterial strain, or its derived component.

Based on prior art it would be natural to think that a strain'scapability of preventing bone loss would be associated with its generaleffect on intestinal health or its anti-inflammatory properties, howeverthe inventors have surprisingly found out that these properties are notpredictive on the efficiency on preventing bone loss. Lactobacillusreuteri ATCC PTA 6475 and Lactobacillus reuteri ATCC PTA 4659 are twoalmost identical strains, which are both anti-inflammatory and improveoverall intestinal health. It is natural to assume that these strainstherefore would have the same effect on bone loss as well. However theinventors have shown that these strains do not have the same impact onpreventing bone loss and based on this observation they have invented anovel way of selecting lactic acid bacterial strains, such as forexample Lactobacillus reuteri that will be effective for treatmentand/or prevention of bone loss.

Lactic acid bacteria specifically selected by the method presentedherein may be administered to humans to prevent bone loss.

L. reuteri ATCC PTA 6475 and ATCC PTA 4659 differ in four SNPs, whichare important for the bacteria's ability to prevent bone loss. TheseSNPs are shown in Walter et al. (Walter et al. Host-microbial symbiosisin the vertebrate gastrointestinal tract and the 30 Lactobacillusreuteri paradigm; PNAS, vol. 108 p. 4645-4652), which is hereby fullyincorporated by reference. For the SNP analysis, sequencing results weremapped onto a reference genome (L. reuteri JCM 1112, GenBank accessionno AP007281, SEQ ID NO: 1). Seven SNPs were found in L. reuteri ATCC PTA4659, and three of them were also found in L. reuteri ATCC PTA 6475 (SNP4 located at bp 567 368, SNP 6 located at bp 968 088, and SNP 8 locatedat bp 1 358 460, referring to the reference genome, L. reuteri JCM 1112,GenBank accession no AP007281, SEQ ID NO: 1). The remaining four uniqueSNPs (for the purpose of the present text, hereinafter called SNP 1, SNP2, SNP 3 and SNP 5, respectively) constitute the genomic differencesbetween L. reuteri ATCC PTA 6475 and L. reuteri ATCC PTA 4659. Said fourSNPs are located at:

-   -   bp 271 391 (SNP 1),    -   bp 453 538 (SNP 2),    -   bp 529 228 (SNP 3), and    -   bp 599 338 (SNP 5),        (referring to the reference genome, L. reuteri JCM 1112, GenBank        accession no AP007281; SEQ ID NO: 1).

SNP 1 is located in a gene coding for a conserved hypothetical protein(L. reuteri JCM 1112: http://www.ncbi.nlm.nih.gov/protein/183224225),SNP 2 is located in a gene coding for a chloride channel protein (L.reuteri JCM 1112: http://www.ncbi.nlm.nih.gov/protein/183224386), SNP 3is located in a gene coding for an ATP synthase gamma subunit (L.reuteri JCM 1112: http://www.ncbi.nlm.nih.gov/protein/183224455) and SNP5 is located in a gene coding for a DNA mismatch repair protein HexB (L.reuteri JCM 1112: http://www.ncbi.nlm.nih.gov/protein/183224511). TheSNPs involved in this invention are the ones that match L. reuteri ATCCPTA 6475, the sequence of which has identical nucleotides as L. reuteriJCM 1112 in the positions of SNP 1, SNP 2, SNP 3 and SNP 5). Listedbelow are the nucleotides that differ between the strains L. reuteriATCC PTA 6475 and 4659:

-   -   SNP 1) a gene coding for a hypothetical protein, where        nucleotide 267 has been changed in ATCC PTA 4659 from a C (as in        ATCC PTA 6475 and JCM 1112) to a T.    -   SNP 2) the gene coding for the chloride channel protein, where        nucleotide 373 has been changed in ATCC PTA 4659 from a G (as in        ATCC PTA 6475 and JCM 1112) to an A.    -   SNP 3) the gene coding for ATP synthase gamma subunit, where        nucleotide 296 has been changed in ATCC PTA 4659 from a G (as in        ATCC PTA 6475 and JCM 1112) to an A.    -   SNP 5) the gene coding for the HexB protein, where nucleotide        1966 has been changed from a C (as in ATCC PTA 6475 and        JCM 1112) to a T.

In the selection method of this invention, strains are sought that, inat least one of these SNPs, harbor the same nucleotides as L. reuteriATCC PTA 6475 for the above mentioned SNPs.

The microbiota plays an important role in bone loss; many patientssuffering from bone loss have a disturbed intestinal microbiota. Lacticacid bacteria that are able to reestablish the normal microbialcommunity in the GI tract are surprisingly more effective in preventingbone loss.

The present invention discloses a unique method of selection, selectingstrains effective for preventing bone loss. The ability to reestablishthe total gut microbial composition is surprisingly also important forthe function in preventing bone loss. The inventors have found out thatstrains capable of reestablishing an altered microbial community tonormal and/or harboring at least one of the four specific SNPs areeffective for preventing bone loss.

The ability to prevent bone loss is unique for certain strains and isnot at all general for all lactic acid bacteria. When selectingeffective strains it is not sufficient to use anti-inflammatory capacityas selection criteria since the inventors clearly show that this effectis not dependent on anti-inflammatory features. L. reuteri ATCC PTA 6475and L. reuteri ATCC PTA 4659 are both anti-inflammatory strains, but L.reuteri ATCC PTA 6475 is much more effective when used for prevention ofbone loss, L. reuteri ATCC PTA 4659 is not selected according to thisinvention. Specific lactic acid bacterial strains selected according tothe present invention may be used for preventing bone loss in generaland the embodiments below are not intended to limit the scope of thisinvention, but to exemplify preferred embodiments.

Vitamin D is crucial to bone health and people with low levels ofvitamin D have lower bone density or bone mass. People that do not getenough vitamin D may lose bone, since vitamin D is required to absorbcalcium. The inventors have seen that an altered microbiota will lead tovitamin D deficiency and bone loss, administration of lactic acidbacteria selected according to the present invention will reestablishthe microbiota and thereby increase the intestinal vitamin D absorptionand restore the levels of vitamin D. It is also an option to combinevitamin D with the selected strains in order to get an even moreefficient method/product for preventing bone loss.

T1D patients suffer from complications such as bone loss. Patientssuffering from T1D will as a result of the condition have an alteredmicrobiota. Administration of lactic acid bacteria selected according tothe present invention will reestablish the microbiota and prevent boneloss.

High bone density during youth and adulthood can help prevent diseaseslike osteoporosis later in life. This is due to the fact that high bonedensity will allow a higher degree of bone loss before reaching a bonedensity within the osteoporosis zone. Thus, it is an object of thepresent invention to prevent bone loss by administering lactic acidbacterial strains, selected according to the present invention, to youngand adult people, this will help individuals to obtain maximum bonedensity to prevent osteoporosis from occurring later in life. Specificlactic acid bacteria selected according to the present inventionprevents bone loss in healthy recipient as well as those suffering frombone loss.

Administration of selected lactic acid bacteria may be combined withhormone replacement therapy Such a combination would make it possible toreduce the amount of hormones and thereby reduce the side effects, suchas reducing the risk of cancer.

Lactic acid bacteria selected for preventing bone loss would preferablybe administered to menopausal women and osteopenic men who are prone todevelop osteoporosis, and administration of selected lactic acidbacteria will prevent bone loss and thus preventing low bone density andosteoporosis.

The inventors have seen that estrogen depletion alters the gutmicrobiota, treatment with lactic acid bacteria selected according tothe present invention will reestablish the microbiota in peoplesuffering from decreased estrogen levels, including but not limited tomenopausal women and women who had hysterectomy, consequently preventingbone loss.

Lactic acid bacterial strains selected according to the presentinvention may also be used to improve fracture repair.

In order to reduce side effects of drugs, such as for examplebisphosphonates and hormone replacement therapy used to treat bone lossit is possible to combine drugs with administration of selected lacticacid bacteria and thereby reduce the dose, which will minimize the sideeffects.

Example 1

Study of L. reuteri ATCC PTA 6475's Ability to Reestablish AlteredMicrobial Communities in Ovx Mice.

There are significant changes in the intestinal microbial communities ofcontrol (non-ovx), ovx and ovx fed by L. reuteri.

Experimental Groups and Tissue Collection.

In order to measure the effects of ovariectomy (ovx) and L. reuteri 6475treatment of ovx mice we compared three experimental groups of animals.Control mice were non-ovx mice that received a vehicle control gavagethree times per week. Ovx mice received a vehicle control gavage threetimes per week. Ovx+L. reuteri 6475 were mice that received 300 μl ofovernight L. reuteri 6475 three times per week for four weeks. At theend of the experiment mice were euthanized and tissue samples from thestomach, duodenum, jejunum, ileum, proximal and distal colons wereisolated and saved for microbial ecology analysis.

DNA Extraction

Murine intestinal tissue was placed in MoBio Ultra Clean Fecal DNA BeadTubes (cat.#12811-100-DBT) containing 360 μl Buffer ATL (Qiagencat.#19076) and lysed on a Mini-Beadbeater-8 (BioSpec Products) for 1minute at full speed. DNA was extracted from murine intestinal tissueusing Qiagen DNeasy Blood and Tissue kit (cat.#69504). The tissue wasfurther disrupted by adding 40 μl proteinase K (Qiagen, cat.#19133) andincubating at 55° C. for 1 hour. DNA was extracted using the QiagenDNeasy Blood and Tissue kit (cat.#69504). DNA yield was quantified usinga Nanodrop 1000.

PCR Amplification

Bacterial 16S sequences were amplified for 454 sequencing from murineintestinal tissue using the V3-V5 barcoded primer set and amplificationprotocol developed by the Broad Institute for the Human MicrobiomeProject. Barcoded forward primers were synthesized by IDT DNATechnologies and the reverse primer was synthesized by Sigma. Barcodedforward primers were diluted to a working concentration of 4 μM in 96well plates; the reverse primer was added to each well to a finalconcentration of 4μM. Triplicate reactions in a 25 μl volume wereprepared containing 400 μg murine intestinal DNA, 2 μl 4 μM primers, and0.15 μl Accuprime HiFi Taq polymerase in 1× Accuprime Buffer II(Invitrogen, cat.#12346086). Reactions were amplified in an EppendorfPro aluminum plate thermal cycler with a 2 minute 95° C. denaturation,followed by 30 cycles of 95° C.×20 sec, 50° C.×30 sec, 72° C.×5 min.

Amplification Product Purification

16S amplification products were purified using Ampure Agencourt XP beads(Beckman Coulter, cat# A63880). First, triplicate reactions for eachsample were combined into 1.7 ml microfuge tubes and Ampure XP beadswere added at a 0.7× volume ratio. After vortexing, the mixed sampleswere incubated for 10 minutes at room temperature then placed on amagnetic stand to separate the beads (Invitrogen, cat.#123-21D). Thebeads were washed according to the manufacturer's protocol with 2 washesof 200 μl of 70% ethanol. The beads were dried at 37° C. for 5 minutes,and DNA was eluted with 20 μl 10 μM Tris buffer/0.1 μM EDTA. The eluentwas separated from the beads on the magnetic stand, transferred to a new1.7 ml microfuge tube, and quantified using the using the Quant-It dsDNAhigh-sensitivity assay kit (Invitrogen, cat# Q33120). Equal amounts ofeach sample were then pooled into one tube for 454 sequencing.

454 Sequencing and Sequence Analysis

454 sequencing was performed using the GS Junior (Roche) using Titaniumchemistry. In addition to the standard filters utilized by the GS Jr. toidentify passed reads we utilized a modified amplicon processingalgorithm to reduce the number of incorrectly discarded sequences. 16SrRNA sequences were aligned by the Ribosomal Database Project staff atMSU to E. coli 16S sequences and trimmed at E. coli 16S nucleotidepositions 617 to 900. Subsequent processing and analysis (includingdiversity metrics) were performed using MOTHUR v.1.21(http://www.mothur.org/wiki/). ANOSIM (analysis of similarity) andprinciple coordinate analysis were performed using the software packagePAST. The accompanying figures and table utilize the Bray-Curtis methodfor measuring the level of dissimilarity between two or more microbialcommunities. In these analyses we chose an operational taxonomic unit(OUT) cutoff of 0.03, which is considered to be viewing the communitiesat the species level. From these data we conclude that in that treatmentof ovariectomized mice with L. reuteri ATCC PTA 6475 causes asignificant shift in the both the ileal and jejunal microbialcommunities, which correlates with improved bone health. (ovx+lacto inthe table 1).

TABLE 1 ANOSIM analysis at species level using Bray-Curtis dissimilaritymatrix, ** indicates statistical significance. Tissue Comparison R Value(p value) Jejunum wt-ovx-ovxlacto 0.3367 (0.0183)* wt-ovx 0.0443(0.3633) wt-ovxlacto 0.6078 (0.0250)* ovx-ovxlacto 0.3297 (0.0712) Ileumwt-ovx-ovxlacto 0.2068 (0.0084)* wt-ovx 0.1710 (0.1180) wt-ovxlacto0.2540 (0.0290)* ovx-ovxlacto 0.2209 (0.0206)*

Three-way comparison of the wild-type, ovx, and ovx treated with L.reuteri showed significant shifts in microbial communities (table 1).These differences are largely driven by substantial shifts incommunities after L. reuteri treatment. Principle coordinate analysis ofmicrobial communities from the wild-type control group (triangle Δ), ovxgroup (circle •) and the ovx group treated with L. reuteri (square ▪)was used to visualize how communities clustered in the jejunum and theileum. FIG. 1 shows that ovx mice treated with L. reuteri form a clusterof communities that is distinct from wild-type and ovx communities inboth the jejunum and ileum. Several OTUs that were classified asClostridriales are the main groups of bacteria that are driving theseparation of the L. reuteri treated Ovx communities from the other twogroups.

Example 2

Study of L. reuteri ATCC PTA 4659 's Ability to Reestablish AlteredMicrobial Communities in Ovx Mice.

The experiment is performed as in example 1, but L. reuteri ATCC PTA4659 is used instead of L. reuteri ATCC PTA 6475.

L. reuteri ATCC PTA 4659 treatment is not able to restore ovxcommunities toward control.

Example 3 Identification of Certain SNPs

Illumina Sequencing of L reuteri Genomes

L. reuteri strains used in this study were ATCC PTA 4659 and 6475 grownin MRS media (Difco) and genomic DNA prepared by using the QiagenGenomic-Tip System. DNA was fragmented by 20 min sonication (130 W) toobtain an average fragment size of 500 bp, then further purified andconcentrated with QIAquick PCR Purification Spin Columns (Qiagen).Treatment to remove 3′ over-hangs and fill in 5′ overhangs resulted inblunt-ended genomic fragments. An adenine residue was added by terminaltransferase to the 3′ end, and the resulting fragments were ligated toSolexa adapters. The products were separated by agarose gelelectrophoresis, and the band between 150 and 200 bp was excised fromthe gel. The DNA fragments were extracted from the agarose slice using aQIAquick Gel Extraction Kit (Qiagen). Adapter-modified DNA fragmentswere enriched by an 18-cycle PCR using Solexa universal adapter primers.The DNA fragment library was quantitated, and then diluted to a 10-nMworking stock for cluster generation. Adapter-ligated fragments (2 nM)were denatured in 0.1 M NaOH for 5 min, then further diluted to a final9 pM concentration in 1 mL of prechilled hybridization buffer, andintroduced onto the Solexa flow cell using the Cluster Station.Following isothermal amplification, clusters were made single-strandedby 0.1 M NaOH denaturation, metered across the flow cell by the SolexaCluster Station. A sequencing primer complementary to one Solexa adapterwas added to prime the single strands of each cluster. Once hybridizedand with excess primer removed by a wash, the flow cell was ready forsequencing. The Solexa Genome Analyzer II was programmed to provide upto 36 sequential flows of fluorescently labeled, 3′-OH blockednucleotides and polymerase to the surface of the flow cell, thusproducing a fixed 36-bp read length. After each base incorporation step,the flow cell surface was washed to remove reactants and then imaged bymicroscope objective. The experiments collected 300 tiled images(“tiles”) per flow cell lane, each containing on average 30,000clusters.

SNP Analysis

The two lanes' sequencing results were mapped onto the reference genomeL. reuteri JCM 1112T (GenBank accession no AP007281) separately. Themapping software Maq version 0.6.6(http://maq.sourceforge.net/maq-man.shtml) was used to perform themapping (default parameters). SNPs were identified and validated by theMAQ software, and classified into coding SNP and intergenetic SNPs.Coding SNPs were identified as synonymous and nonsynonymous. The SNPswere finally verified by PCR amplification of the surrounding region,followed by Sanger sequence determination.

Example 4 Method of Selection of Strains

The selection of strains effective for prevention of bone loss is basedon the ability to restore altered microbial communities. Based on theresults of examples 1 and 2, L. reuteri ATCC PTA 6475 is selected basedon the fact that this strain has the ability to restore alteredmicrobial communities. L. reuteri ATCC PTA 4659 is not selected based onthe results of example 2.

Example 5 Method of Selection of Strains

The selection of strains effective for prevention of bone loss is basedon the presence of certain SNPs. As a consequence of the results ofexample 3, L. reuteri ATCC PTA 6475 is selected, since it harbors all ofthe four sought SNPs. Due to the lack of these SNPs L. reuteri ATCC PTA4659 is not selected.

Example 6 Method of Selection of Strains

The selection of strains effective for prevention of bone loss is basedon example 3 and 4 and 5, strains harboring at least one of the foursought SNPS as well as the capacity to restore altered microbialcommunities is selected. Based on these criteria L. reuteri ATCC PTA6475 is selected.

Example 7

L. reuteri ATCC PTA 6475 Suppresses Ovx Induced Bone Loss

In this study ovariectomized (ovx) BALB/c mice were used as a mousemodel for bone loss. Mice (12 weeks old) were ovariectomized and dividedinto two groups were the first group was treated with L. reuteri ATCCPTA 6475 three times a week during four weeks. BALB/c that had not beenovariectomized were used as a control group. Distal femur bone volumefraction (BV/TV) and bone TRAP5 RNA (relative to HPRT) were measured.Mice treated with L. reuteri ATCC PTA 6475 showed the same bone volumefraction as the control group. Further it was to be seen that TRAP5 (amarker of osteoclast function) is returned to baseline (control group)upon L. reuteri ATCC PTA 6475 treatment.

FIG. 2 shows that the suppression of bone loss by L. reuteri ATCC PTA6475 is nearly 100% and that the expression of TRAP5 is returned tobaseline.

Example 8

The Selected L. reuteri ATCC PTA 6475 is Superior to the Non-Selected L.reuteri ATCC PTA 4659 in Suppressing Bone Loss.

In this experiment we gavaged animals three times per week with the L.reuteri ATCC PTA 6475 and L. reuteri ATCC PTA 4659 strains while alsoproviding the strains continuously in the drinking water for 28 days.Distal femur bone volume fraction (BV/TV) was measured by μCT. L.reuteri ATCC PTA 6475 suppressed bone loss and was indistinguishablefrom control mice (FIG. 3). L. reuteri ATCC PTA 4659 did not suppressbone loss to a sufficient level that reached statistical significance(p<0.01). L. reuteri ATCC PTA 4659 is not as effective as the selectedstrain L. reuteri ATCC PTA 6475.

1. A method for preventing or treating bone loss, comprisingadministering to an individual in need thereof a lactic acid bacterialstrain comprising a nucleic acid sequence having at least 99% identityto the nucleic acid sequence of the genome of L. reuteri JCM 1112 (SEQID NO:1), and wherein the lactic acid bacterial strain harbors anucleotide relative to the genome of Lactobacillus reuteri JCM 1112 (SEQID NO: 1) in at least one of the following four positions: C in basepair 271 391, G in base pair 453 538, G in base pair 529 228, and C inbase pair 599
 338. 2. The method of claim 1, wherein the lactic acidbacterial strain harbors a nucleotide relative to the genome ofLactobacillus reuteri JCM (SEQ ID NO:1) in at least two of the followingfour positions: C in base pair 271 391, G in base pair 453 538, G inbase pair 529 228, and C in base pair 599
 338. 3. The method of claim 1,wherein the lactic acid bacterial strain harbors a nucleotide relativeto the genome of Lactobacillus reuteri JCM (SEQ ID NO:1) in at leastthree of the following four positions: C in base pair 271 391, G in basepair 453 538, G in base pair 529 228, and C in base pair 599
 338. 4. Themethod of claim 1, wherein the lactic acid bacterial strain harbors anucleotide relative to the genome of Lactobacillus reuteri JCM (SEQ IDNO:1) in all of the following four positions: C in base pair 271 391, Gin base pair 453 538, G in base pair 529 228, and C in base pair 599338.